CN1341713A - Extraction method of crataegus fruit SOD - Google Patents
Extraction method of crataegus fruit SOD Download PDFInfo
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- CN1341713A CN1341713A CN 01136564 CN01136564A CN1341713A CN 1341713 A CN1341713 A CN 1341713A CN 01136564 CN01136564 CN 01136564 CN 01136564 A CN01136564 A CN 01136564A CN 1341713 A CN1341713 A CN 1341713A
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- sod
- ammonium sulfate
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Abstract
The preparation method of crataegus fruit SOD crude enzyme mainly includes the following steps: crushing crataegus fruit, centrifugalizing to obtain crude extract, using 30% ammonium sulfate to make precipitation treatment, and adopting membrane system to make filtration and obtain filtrate, using 90% ammonium sulfate to make the filtrate undergo the process of precipitation treatment, then dissolving the precipitate in buffer solution, using Sephadex G-25 to desalt to obtain crude enzyme extract, adding the crude enzyme extract into Sephadex G-100 column to make column chromatographic treatment, adopting full-automatic fraction collecting system to collect eluent, and combining collected liquors with high SOD activity, further dialysis, freezing and drying so as to obtain high enzyme activity SOD crude enzyme preparation.
Description
Technical field:
The invention belongs to bioengineering field, relate in particular to a kind of extractive technique of SOD enzyme.
Background technology:
The SOD enzyme is one of biological important protective enzyme, and energy enhancing body anti-adversity ability is anti-aging.Existing document shows in a lot of organisms and has the SOD enzyme, extracts and utilizes the SOD enzyme to become one of focus of current science and technology research.Number of patent application 97108883 discloses a kind of method of extracting SOD from earthworm, the steps include: to wash earthworm, purifies, and percolating solution is produced the SOD mother liquor, coarse filtration, the centrifugal supernatant liquor of producing, ultrafiltration, reverse osmosis concentration, lyophilize etc.Number of patent application 86100551 discloses the method for purification of a kind of superoxide dismutase (SOD), and it is that animal livers homogenate substep heating and dialysis precipitation under the condition of cupric salt are removed impurity, extracts SOD; Adopt fraction precipitation of ammonium sulphate, remove impurity elimination egg hundred, collect the protein that contains SOD, through dialysis, concentrated, dry, can obtain than vigor is the SOD of 2789 unit/milligrams again.Number of patent application 91101832 discloses a kind of production of soybeam superoxides dismutase, it is to be raw material production soybeam superoxides dismutase (soybean SOD) with soybean (beans), and its production method is that soybean is passed through successively: make operations such as swelled soybean, obtain soy milk, tone pitch are handled, centrifugal treating gets supernatant liquor, heating and filtering, ultrafiltration and concentration, drying and obtain SOD.But above-mentioned patent application is all because of technical measures or the raw material chosen is improper implements.Hawthorn ranges Rosaceae hawthorn, is perennial deciduous fruit tree.Through mensuration, contain higher SOD activity in the hawthorn fruit, but its extraction process is not carried out deep research so far, by retrieval, find no about from hawthorn fruit, extracting the report of SOD enzyme.
Summary of the invention:
The applicant proposes a kind of production technique of extracting the thick enzyme of crataegus fruit SOD by testing repeatedly for many years and studying.The thick enzyme of crataegus fruit SOD of the present invention can obtain by ammonium sulfate precipitation method or membrane separation process.The present invention implements like this:
1, raw materials pretreatment
New fresh haw berry is smashed pulping to pieces by tissue mashing machine, places the centrifugal 10-30 of 4000-6000rpm whizzer minute, abandons the precipitation residue, obtains supernatant liquor as crude extract.
2, ammonium sulfate precipitation method extracts crataegus fruit SOD
With crude extract 30% ammonium sulfate precipitation, adopt the film system filtration to obtain filtrate; The filtrate that obtains is used 90% ammonium sulfate precipitation, precipitation is dissolved in the damping fluid SephadexG-25 desalination then; Thick zyme extract after the desalination joined in the Sephadex G-100 post carry out column chromatography, adopt full-automatic distribution gathering system to collect elutriant, merge the high collection liquid of SOD vigor; Further dialysis, lyophilize obtains high enzyme SOD crude zyme preparation alive.
Other method of the present invention is that membrane ultrafiltration extracts crataegus fruit SOD, and its extraction step is:
1, raw materials pretreatment
New fresh haw berry is smashed pulping to pieces by tissue mashing machine, places the centrifugal 10-30 of 4000-6000rpm whizzer minute, abandons the precipitation residue, obtains supernatant liquor as crude extract.
2, membrane ultrafiltration extracts crataegus fruit SOD.But 60,000 membrane ultrafiltration (tubular fibre, flat sheet membrane and rolled film) with crude extract by molecular weight cut-off, after the crude extract ultrafiltration, acquisition contains the clear liquid of molecular weight 60,000 following materials, but clear liquid is that 10,000 membrane ultrafiltration (tubular fibre, flat sheet membrane and rolled film) concentrates by molecular weight cut-off again, obtain the concentrated solution of molecular weight 1～60,000, further lyophilize can obtain the SOD crude zyme preparation.
The present invention compares than prior art has following typical advantages:
1, extraction yield height.Its extraction yield of method of the present invention is up to 80%.
2, the SOD enzyme is active high.
3, raw material is easy to get, and is with low cost.
Description of drawings:
Fig. 1 is a process flow sheet of the present invention
The working of an invention mode
Embodiment 1: ammonium sulfate precipitation method extracts crataegus fruit SOD:
1, raw materials pretreatment
New fresh haw berry is smashed pulping to pieces by tissue mashing machine, places the centrifugal 10-30 of 4000-6000rpm whizzer minute, abandons the precipitation residue, obtains supernatant liquor as crude extract.
2,30% ammonium sulfate precipitation
Crude extract is added ammonium sulfate, make that ammonium sulfate reaches 30% saturation ratio in the crude extract.Stirred 55 minutes down in room temperature (23-25 ℃), adopt the film system filtration to obtain filtrate.
3,90% ammonium sulfate precipitation
In the filtrate behind 30% ammonium sulfate precipitation, adding ammonium sulfate makes its saturation ratio reach 90%, at room temperature stirred 60 minutes, placed the centrifugal 20-40 of 10000-15000rpm whizzer minute, precipitation is dissolved in the 5mM pH7.8 phosphoric acid buffer of minimum (containing 2mM EDTA), uses above-mentioned damping fluid and SephadexG-25 desalination then.
4, Sephadex G-100 column chromatography
Sephadex G-100 post is used the potassium phosphate buffer balance of pH7.8,5mM in advance; Thick zyme extract after the desalination is joined in the post, and use above-mentioned buffer solution elution.Adopt full-automatic distribution gathering system to collect elutriant, merge the high collection liquid of SOD vigor.
Embodiment 2: membrane ultrafiltration extracts crataegus fruit SOD
After hawthorn fruit 100kg smashed to pieces through tissue, centrifugal removal impurity stayed supernatant liquor 30L as crude extract, enzyme 156U/ml alive; But is 60,000 membrane ultrafiltration (tubular fibre, flat sheet membrane and rolled film) with clear liquid by molecular weight cut-off, after the clear liquid ultrafiltration, obtains to contain the clear liquid 27L of molecular weight 60,000 following materials, enzyme work 140U/ml, the rate of recovery 80%; But the 27L clear liquid is that 10,000 membrane ultrafiltration (tubular fibre, flat sheet membrane and rolled film) concentrates by molecular weight cut-off again, obtains the concentrated solution 2.5L of molecular weight 1～60,000, the enzyme 1200U/ml that lives, the rate of recovery 79%.Further lyophilize can obtain the SOD crude zyme preparation.
Embodiment 3: ammonium sulfate precipitation method extracts crataegus fruit SOD
After hawthorn fruit 100kg smashed to pieces through tissue, centrifugal removal impurity stayed supernatant liquor 30L as crude extract, enzyme 156U/ml alive; With crude extract 30% ammonium sulfate precipitation, adopt the film system filtration to obtain filtrate; The filtrate that obtains is used 90% ammonium sulfate precipitation, and precipitation is dissolved in the damping fluid, and SephadexG-25 desalination then obtains thick zyme extract 24L; Thick zyme extract after the desalination joined in the Sephadex G-100 post carry out column chromatography, adopt full-automatic distribution gathering system to collect elutriant, merge the high collection liquid of SOD vigor; Further dialysis, lyophilize obtains high enzyme SOD crude zyme preparation alive 120.3g, and enzyme is lived and is 30085U/g.By the SOD in this method extraction hawthorn, the rate of recovery is 77.3%.
The mensuration of embodiment 4:SOD
The measuring method of SOD adopts the NBT photochemical method, and is specific as follows:
(1) equipment: 752 type spectrophotometers; Desk centrifuge; Illumination box; Refrigerator; 10ml small beaker etc.
(2) reagent: chlorination nitrate blue tetrazolium (NBT)---the Shanghai preparation factory that advances produces
L-methionine(Met) (L-Met)---Kangda Amino-acid Factory of Shanghai produces
Ethylenediamine tetraacetic acid (EDTA) (EDTA)---Beijing Chemical Plant produces
Riboflavin (VB
2)---the chemical plant produces in the west, Beijing
(3) preparation of reaction solution
The preparation of NBT reaction solution: preparation pH7.8, the phosphoric acid buffer 1000ml of 50mmol/L, the L-Met that adds 1.933mg, after treating to dissolve fully, put into 45.8mg NBT, 0.47mg riboflavin (be made into 0.47mg/ml solution, draw 1ml and add) and 29.0mgEDTA, solution final concentration: Met is 13mmol/L; NBT is 6.3 * 10
-3Mmol/L; Riboflavin is 1.3 * 10
-3Mmol/L; EDTA is 0.1mmol/L, places brown reagent bottle, with the shading of black cloth cover, can preserve 1 month in 4 ℃ of refrigerators.
(4) measuring method
Draw 30 μ l samples, put into 10ml transparent glass small beaker, add the 3mlNBT reaction solution, mixing is in 28 ℃ of SOD photochmeical reaction chambers, 2 * 15W fluorescent lamp illumination 20 minutes (illumination is 4000 Luxs), measure absorbancy at the 560nm place, make blank, establish 3 repetitions for every group with the NBT reaction solution, whole mensuration process is all carried out under lucifuge or low light condition except that photochmeical reaction.Contrast as test with the standard enzyme of U.S. ＂ Sigma ＂ company, Bao'an, Shanghai company and the SOD oral liquid of Laolaifu Pharmaceutical Co., Guizhou's production in the test process simultaneously.
(5) calculate
In the formula: CK---the blank transparence
N---product extension rate
A---sample transparence
Enzyme unit: U/g (ml) alive
Claims (2)
1, a kind of ammonium sulfate precipitation method extracts the crataegus fruit SOD enzyme, it is characterized in that extraction step is:
(1) raw materials pretreatment
New fresh haw berry is smashed pulping to pieces by tissue mashing machine, places the centrifugal 10-30 of 4000-6000rpm whizzer minute, abandons the precipitation residue, obtains supernatant liquor as crude extract;
(2) 30% ammonium sulfate precipitations
Crude extract is added ammonium sulfate, make that ammonium sulfate reaches 30% saturation ratio in the crude extract, stirred 55 minutes down, adopt the film system filtration to obtain filtrate in room temperature (23-25 ℃);
(3) 90% ammonium sulfate precipitations
In the filtrate behind 30% ammonium sulfate precipitation, adding ammonium sulfate makes its saturation ratio reach 90%, at room temperature stirred 60 minutes, placed the centrifugal 20-40 of 10000-15000rpm whizzer minute, precipitation is dissolved in the 5mM pH7.8 phosphoric acid buffer of minimum (containing 2mM EDTA), uses above-mentioned damping fluid and SephadexG-25 desalination then;
(4) Sephadex G-100 column chromatography
Sephadex G-100 post is used the potassium phosphate buffer balance of pH7.8,5mM in advance; Thick zyme extract after the desalination is joined in the post, and use above-mentioned buffer solution elution, adopt full-automatic distribution gathering system to collect elutriant, merge the high collection liquid of SOD vigor.
2, a kind of membrane ultrafiltration extracts the crataegus fruit SOD enzyme, it is characterized in that following step:
(1) raw materials pretreatment
New fresh haw berry is smashed pulping to pieces by tissue mashing machine, places the centrifugal 10-30 of 4000-6000rpm whizzer minute, abandons the precipitation residue, obtains supernatant liquor as crude extract.
(2) membrane ultrafiltration
But is 60,000 membrane ultrafiltration with crude extract by molecular weight cut-off, after the crude extract ultrafiltration, obtains to contain the clear liquid of molecular weight 60,000 following materials, but clear liquid is that 10,000 membrane ultrafiltration concentrates by molecular weight cut-off again, obtain the concentrated solution of molecular weight 1～60,000, lyophilize can obtain the thick enzyme of SOD.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01136564 CN1341713A (en) | 2001-10-18 | 2001-10-18 | Extraction method of crataegus fruit SOD |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01136564 CN1341713A (en) | 2001-10-18 | 2001-10-18 | Extraction method of crataegus fruit SOD |
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| CN1341713A true CN1341713A (en) | 2002-03-27 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106987565A (en) * | 2017-05-22 | 2017-07-28 | 大连大学 | A kind of marine low temperature superoxide dismutase extraction and separation process |
| CN107213029A (en) * | 2017-05-22 | 2017-09-29 | 大连大学 | A kind of moisturizing toner for adding low-temperature superoxide dismutase and preparation method thereof |
-
2001
- 2001-10-18 CN CN 01136564 patent/CN1341713A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106987565A (en) * | 2017-05-22 | 2017-07-28 | 大连大学 | A kind of marine low temperature superoxide dismutase extraction and separation process |
| CN107213029A (en) * | 2017-05-22 | 2017-09-29 | 大连大学 | A kind of moisturizing toner for adding low-temperature superoxide dismutase and preparation method thereof |
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